Search

An increased angiogenesis has been documented in bone marrow and lymph nodes of patients with chronic lymphocytic leukemia (CLL). There is accumulating evidence that circulating endothelial cells (CECs) play an important role in angiogenesis. The aim of our study was to compare the number of CECs in peripheral blood of CLL patients and healthy donors and to correlate these numbers with bone marrow microvessel density (MVD) and known prognostic factors in CLL. Proportions of resting CECs (rCECs), activated CECs (aCECs), apoptotic CECs (apoCECs) and circulating precursor endothelial cells (CPECs) were estimated by flow cytometry in 104 untreated CLL patients and 29 healthy blood donors. The MVD was analysed in the bone marrow biopsy in 21 CLL patients and 11 controls using the 'hot spot analysis' of vessels' density (×200 HPS). We found significantly higher numbers of CPECs, rCECs, aCECs and apoCECs in CLL patients than in healthy controls (p < 0.001 for all comparisons). Furthermore, the rCECs number was higher in advanced versus low clinical stage CLL (median 17.5 cells/µL vs. 13.5 cells/µL, p = 0.05). The MVD was significantly higher in the bone marrow of CLL patients when compared with controls (p = 0.016). However, we did not find any correlation between MVD and different CECs populations. In conclusion, the number of CECs is increased in CLL and correlates with stage of the disease, but does not seem to directly reflect the intensity of neovascularisaton in the bone marrow. [ABSTRACT FROM AUTHOR]

Abstract: Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31+, CD146+, CD45−, 7-amino-actinomycin-D (7AAD)− events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 106) to ensure statistical analysis, and (4) use of 10 µm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r 2 =0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r 2 =0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0–15) in healthy individuals and 15.0/ml (range, 0–179) in patients with metastatic carcinoma (p <0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients. [Copyright &y& Elsevier]

Abstract: Background: Metronomic chemotherapy (MC) is the administration of chemotherapy at doses below the maximal tolerated dose on a frequent schedule of administration, with no prolonged drug-free breaks. Objective: The aim of this research was to assess the effectiveness and tolerance of a metronomic etoposide/ cyclophosphamide/celecoxib regimen in children and adolescents with refractory cancer. Methods: This retrospective, single-center study evaluated the use of MC with etoposide 25 mg/m2.d-1 (days 1-14), cyclophosphamide 25 mg/m2 d-1 (days 15-28), and celecoxib 100 to 400 mg/d (days 1-28), in children with refractory, or high-risk relapsing, cancer. Adverse events were determined through laboratory analyses and investigator observations. Results: From January 2005 to December 2007, 17 children and adolescents were treated. The best responses observed were stabilizations of the disease that lasted over 20 weeks in 7 patients (41%). Most importantly, in 4 patients (24%) antalgic treatment could be transiently diminished or stopped, and in 1 patient (6%) oxygen support could be stopped for several weeks. Four grade IV platelet toxicities were noted in 3 patients; 2 grade IV anemia occurred in 2 patients (who had platelet and red blood cell transfusions before initiation of treatment); and 1 patient had grade III neutropenia. No other grade III or IV toxicities were noted. Grade II alopecia and stomatitis were observed in 1 patient and grade II vomiting was observed in 2 patients. One patient with meningeal carcinomatosis developed bilateral subdural hematoma for which the role of MC could not be ruled out. Circulating endothelial cells were elevated in 3 out of 3 patients in whom they were quantified and who were progressing while under MC. Conclusion: The MC regimen we report here was associated with disease stabilization without major toxicities. This assessment of MC in children and adolescents warrants further studies. [Copyright &y& Elsevier]

Increased circulating endothelial cells (CECs) have been observed in patients with vascular injury associated with acute myocardial infarction, pulmonary hypertension, and congestive heart failure. Decreased circulating endothelial progenitor cells (EPCs) have been observed in patients with risk factors for cardiovascular disease. Obstructive sleep apnea (OSA) is associated with increased risk of cardiovascular disease and endothelial dysfunction. Subjects were recruited from patients referred for overnight polysomnograms; 17 subjects had OSA and 10 control subjects did not have OSA. All subjects lacked vascular disease and risk factors for vascular disease. Peripheral blood was obtained from fasting subjects in the morning, following sleep studies. CECs and EPCs were quantified using magnetic bead separation with UV epifluorescence microscopy and flow cytometry immunophenotyping, respectively. Cell counts and demographic variables were compared using unpaired t tests. Regression analysis was performed comparing cell counts with the apnea-hypopnea index (AHI) and nadir SaO2. Subjects with OSA and controls did not differ significantly in terms of age and body mass index. Subjects with OSA had higher AHI, lower nadir SaO2, and greater sleepiness (Epworth Sleepiness Scale scores). There were no significant differences in CEC (7.0 ± 1.5 vs. 4.9 ± 0.9, p > 0.05) or EPC (1077 ± 318 vs. 853 ± 176, p > 0.05) between controls and OSA cases, respectively. In this small study, we found no differences in CECs or circulating EPCs between patients with OSA and controls. OSA may not be associated with these markers of vascular endothelial cell injury in patients with no concomitant vascular disease. [ABSTRACT FROM AUTHOR]

Circulating endothelial cells (CECs) are associated with neoangiogenesis in various malignant disorders. Using flow cytometry, we studied CECs in 128 patients with myelodysplastic syndrome (MDS). MDS patients had higher CEC levels than controls (P<0.001), and an inverse relationship was found between CECs and international prognostic scoring system risk (r=−0.55, P<0.001). There was a positive correlation between marrow microvessel density and CECs, low-risk patients showing the strongest association (r=0.62, P<0.001). We calculated a progenitor-to-mature CEC ratio, which was higher in MDS patients than in healthy subjects (P<0.001), the highest values were found at diagnosis. CECs assessed by flow cytometry positively correlated with the ability to produce endothelial colony-forming cells in vitro (ECFCs; r=0.57, P=0.021), which was significantly higher in MDS patients than in controls (P=0.011). Fluorescence in situ hybridization analysis showed that a variable proportion of CECs (from 40 to 84%) carried the same chromosomal aberration as the neoplastic clone, while endothelial cells isolated from in vitro assays were negative. This study suggests that CECs reflect the abnormal angiogenesis found in MDS, especially in the early stages of the disease. The increased number of functional endothelial progenitor cells in MDS strengthens the rationale for therapeutic interventions aimed at restoring a normal interaction between hematopoietic progenitors and marrow microenvironment.Leukemia (2008) 22, 530–537; doi:10.1038/sj.leu.2405069; published online 20 December 2007 [ABSTRACT FROM AUTHOR]

Preclinical studies demonstrate anti-angiogenic activity of low doses of chemotherapy; selective cox-2 inhibitors are also inhibitors of angiogenesis. Animal data indicates the presence of circulating endothelial cells (CEC), tumor-derived activated endothelial cells (AEC) and endothelial cell progenitors (ECP). Bone marrow-derived ECP have been shown to be incorporated into tumor vasculature. We conducted two combination Phase I studies of celecoxib with either cyclophosphamide or etoposide. Exploratory correlative studies were performed to evaluate the detectability of CEC, AEC and ECP in patients treated with these anti-angiogenic combinations. Patients were treated with oral cyclophosphamide at 50 mg daily or etoposide at 50 mg daily. Celecoxib was given at 400 mg twice daily. Blood samples were collected on days 0, 7, 28 and monthly until disease progression. Blood from healthy volunteers was collected on days 0 and 28. Peripheral mononuclear cells (PMNC) were isolated and stained with fluorescent antibodies and analyzed utilizing 5-color flow cytometry. Forty-four heavily pretreated patients (20 F; 24 M) with various solid tumors were enrolled. Median age was 65 (23-72). Therapy was well tolerated. No responses were seen. Six patients had stable disease for at least 16 weeks. The longest duration on therapy is 420 days in a patient with metastatic thymoma. Pre-therapy CEC were detected in cancer patients and normal controls with mean concentrations of 0.47 cells/uL and 0.14 cells/uL, respectively. Mean ECP in patients and controls were 0.09 cells/ uL and 0.03 cells/uL, respectively. No AEC were detected. No consistent changes were seen in CEC or ECP during therapy. The combinations of oral cyclophosphamide or etoposide at 50 mg daily with celecoxib at 400 mg twice daily are well tolerated with occasional prolonged disease stabilizations observed. CEC and ECP are detectable in cancer patients but their levels did not change significantly during therapy with our regimen. Further evaluation of CEC and ECP in patients treated with clinically more active anti-angiogenic therapies would be of interest. [ABSTRACT FROM AUTHOR]

The circulating endothelial cells (CEC) are proposed to be a noninvasive marker of angiogenesis. Recent data suggest that endothelial cells may enhance the survival and proliferation of leukemic blasts and mediate chemotherapy resistance in acute myeloid leukemia (AML). We analyzed CEC count by the four-color flow cytometry in AML and healthy subjects. We evaluated the kinetics of mature CEC, both resting (rCEC) and activated (aCEC), as well as progenitor (CEPC) and apoptotic CEC (CECAnnV+) in AML patients treated with standard chemotherapy and their influence on response to treatment and overall survival. We found significantly higher numbers of aCEC, rCEC, CEPC, and CECAnnV+ in AML patients than in healthy controls. The elevated CEPC and absolute blood counts in peripheral blood as well as the low CECAnnV+ number were associated with higher probability of induction treatment failure. aCEC, rCEC, CEPC, and CECAnnV+ counts determined in complete remission (CR) were significantly lower than those found at diagnosis. In those CR patients, a significant decrease in the CEC count and increase in the number of CECAnnV+ were observed already 24h after the first dose of chemotherapy. In refractory AML, the aCEC, rCEC, CEPC, and CECAnnV+ counts assessed before and after induction chemotherapy did not differ significantly, and a significant decrease in CEC count and increase in CECAnnV+ number were noted only after the last dose of chemotherapy. The number of CEC is significantly higher in AML patients than in healthy subjects and correlates with response to treatment. The evaluation of CEC kinetics and apoptotic profile may be a promising tool to select AML patients with poor response to chemotherapy who may benefit from antiangiogenic therapies. [ABSTRACT FROM AUTHOR]

Background. The quantification of circulating endothelial cells (CECs) in whole blood is an increasingly recognized index of endothelial damage/dysfunction. Abnormal CECs have been linked to the severity of coronary artery disease (CAD). Objective. We assessed the relationship of CECs to other markers of endothelial dysfunction (von Willebrand factor (vWF) and soluble E-selectin (sEsel)) during exercise stress testing (EST) in a cohort of patients with suspected CAD. Methods. We studied a cohort of patients referred to our chest pain clinic with a history of exertional chest pain. Treadmill EST was performed, using a full Bruce exercise protocol. Blood for CECs (immunobead method), vWF and sEsel (both ELISA) were collected immediately before (pre-exercise), immediately following exercise, and at 30 minutes post-EST. Results. We studied 31 patients (84% male; mean (SD) age 58.4 (9.8) years). Of the entire cohort, 14 patients (45.2%) had a positive EST. Exercise led to significant increases in levels of CECs, sEsel, vWF, white blood cells (WBC), heart rate, mean and systolic blood pressure compared with base-line (all P<0.05). There was a significant correlation between the change (Δ (immediate post-pre-exercise)) in CECs and ΔvWF (r = 0.45; 95% CI 0.11-0.69: P = 0.01) and ΔsEsel (r = 0.41; 0.05-0.7: P = 0.02), as well as between ΔvWF and ΔsEsel (r = 0.55; 0.25-0.76: P = 0.001). Neither absolute nor ΔCEC counts were predictive of exercise work-load/functional capacity, nor the presence of positive EST results. Conclusion. EST led to a significant increase in endothelial markers (CECs, vWF, and sEsel) compared with base-line levels. The rise in CECs correlated with the increases in other endothelial markers, but was not related to the either exercise work-load/capacity or to the presence of a positive EST. [ABSTRACT FROM AUTHOR]

High altitude and hypoxia are known to induce polycythemia, pulmonary hypertension, and vascular remodeling. The authors investigated a number of blood cell populations in 15 mountain trekkers before and after 12 days spent at >3000 m. Red blood cell and platelet count increased, whereas circulating hematopoietic stem cell (enumerated as CD34bright cells), circulating endothelial cell (CEC) and circulating endothelial progenitor (CEP) count significantly decreased. In particular, the authors observed a decrease in the count of viable CECs, and a decrease in the circulating levels of RNA of the endothelial-specific gene VE-cadherin, whereas the fraction of apoptotic/necrotic CECs was stable. These data suggest a unique pattern of modulation of surrogate markers of vascular remodeling induced by exposure to hypobaric hypoxia. [ABSTRACT FROM AUTHOR]

The article presents a study on the effects of percutaneous coronary intervention (PCI) on peripheral venous blood that circulates endothelial cells and plasma indices of endothelial damage/dysfunction. The effects of diagnostic coronary angiography among comparable patient groups and healthy control subjects were investigated. A significant increase in three endothelial markers with elective PCI were observed.

Sedentary behavior has deleterious effects on the cardiovascular system, including reduced endothelial functions. A 2-mo bed rest study in healthy women [women international space simulation for exploration (WISE) 2005 program] presented a unique opportunity to analyze the specific effects of prolonged inactivity without other vascular risk factors on the endothelium. We investigated endothelial properties before and after 56 days of bed rest in 8 subjects who performed no exercise (control group: No-EX) and in 8 subjects who regularly performed treadmill exercise in a lower body negative pressure chamber as well as resistance exercise (countermeasure group, EX). A functional evaluation of the microcirculation in the skin was assessed with laser Doppler. We studied endothelium-dependent and -independent vasodilation using iontophoresis of acetylcholine and sodium nitroprusside, respectively. We also measured circulating endothelial cells (CECs), an index of endothelial damage. In the No-EX group, endothelium-dependent vasodilation was significantly reduced (35.4 ± 4.8% vs. 24.1 ± 3.8%, P < 0.05) by bed rest with a significant increase in the number of CECs (3.6 ± 1.4 vs. 10.6 ± 2.7 ml-1, P < 0.05). In the EX group, endothelium-dependent vasodilation and number of CECs were preserved. Our study shows that in humans prolonged bed rest causes impairment of endothelium-dependent function at the microcirculatory level, along with an increase in circulating endothelial cells. Microcirculatory endothelial dysfunction might participate in cardiovascular deconditioning, as well as in several bed rest-induced pathologies. We therefore conclude that the endothelium should be a target for countermeasures during periods of prolonged deconditioning. [ABSTRACT FROM AUTHOR]

The article reports on the results of a study of the relationship between endothelial damage/dysfunction, arterial stiffness and their association with predicted risk of future cardiovascular death among patients with hypertension. A description of study methods is presented. The study concluded that there is a consistent association between circulating endothelial cells, arterial stiffness and the predictive risk of cardiovascular death among a group of patients with high-risk hypertension.

Accumulating evidence suggests that angiogenesis may play a key role in the pathogenesis of leukaemic disorders. Several studies have shown that bone marrow-derived endothelial cells (EC) may contribute to tumour angiogenesis and that in the peripheral blood of cancer patients there is an increased amount of circulating ECs (CECs) that may participate to new vessel formation. In this report, we showed that, in seven acute myeloid leukaemia (AML) patients with known cytogenetic abnormalities, CEC levels were significantly increased in comparison with controls and that a significant proportion of these CECs carried the same chromosomal aberration as blast cells (20–78%, mean value 42.1% of CECs). Most of CECs (mean value 74.4%) displayed immunophenotypic features of endothelial progenitor cells as they expressed CD133, a marker gradually lost during EC differentiation and absent on mature EC. These findings suggest a possible direct contribution of AML-related CECs to tumour vasculogenesis and possibly to the spreading and progression of the disease. [ABSTRACT FROM AUTHOR]

Chemotherapy continues to be the main therapeutic approach in the treatment of hematological malignancies including acute leukemia. Generally, chemotherapy is used to eliminate cancer cells and to restore normal bone marrow function. Simultaneous action of cytostatic drugs on bone marrow angiogenesis decreases the formation of new capillaries and improves therapeutic effect. However, chemotherapeutic agents may also be cytodestructive for cellular elements of other tissues, particularly the vascular endothelium, which can lead to various cardiovascular complications. In this work, we studied the effects of 2 cytostatic drugs, cytosine arabinoside (ara-C) and daunorubicin (DNR), on cultured human vascular (i.e., umbilical) endothelial cells (ECs). Ara-C and DNR were added to cultured cells at concentrations ranging from 1 ng/mL to 100 μg/mL. Drug effects were studied using phase-contrast microscopy, cell viability tests, BRDU incorporation, immunohistochemistry, flow cytometry, and cell cloning. At various concentrations, ara-C and DNR are able to induce morphological and functional changes in cultured cells related to either cytostatic or cytotoxic action. Moreover, ara-C-treated cultured cells displayed significant disturbances in cell adhesion molecule expression and interaction with blood leukocytes. Preliminary data obtained on acute leukemia patients undergoing standard cytostatic therapy (“7+3” regimen) have shown that concentration of the circulating ECs was significantly increased compared with the control group and could be as high as 500–1500 cells/mL of blood. Results obtained suggest that anticancer chemotherapy may induce systemic damage of vascular endothelium related to massive cell loss and (or) alterations of endothelial function. [ABSTRACT FROM AUTHOR]

Abstract: Circulating endothelial cells (CECs) were first described over 30 years ago in smears of peripheral blood. Since then, more sophisticated techniques for CEC isolation have become available. In particular, immunomagnetic isolation and fluorescence-activated cell sorting (FACS) have been employed with success. We provide a short historical perspective and a comprehensive review on the subject. We review isolation and enumeration of CECs with an emphasis on CD146-driven immunomagnetic isolation and FACS. We describe, in great detail, advantages and pitfalls of both approaches and compare their specificity. Moreover, we provide a comprehensive list of clinical studies in this field and describe the possible clinical use of CECs. We also describe the phenotype of these cells and list typical surface markers. In addition, we review the phenotype of CECs and discuss mechanisms of detachment. We speculate about potential interactions between CECs and other cell subsets. We also describe other serum markers of endothelial damage and compare CECs with these markers. Finally, we highlight differences between circulating endothelial cells and endothelial progenitor cells. In summary, CECs must now be regarded as a sensitive and specific marker of endothelial damage. We emphasize that use of CECs in a clinical setting is on the horizon and pathogenetic clues may also be obtained. [Copyright &y& Elsevier]

Abstract: Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) are two populations of recently discovered endothelioid cells present in the blood. The former are thought to arise from the intima, the latter from the bone marrow. However, it is becoming clear that these are not in fact homogenous populations (e.g. differing degrees of apoptosis, necrosis and viability, differing expression of monocyte markers) but do in fact represent more than one species of endothelioid cell. Thus whilst originally defined by different criteria (e.g. CD146 by immunobeads, CD34 by flow cytometry) and the perception of independence, there is also growing evidence of some degree of commonality, i.e. some cells co-expressing CD146 and CD34. Furthermore, relationships between these two cells types and, for example, plasma and physiological indicators of vascular damage, and the risk factors for atherosclerosis, suggest a potential role for these cells in the pathophysiology of this disease, possibly as markers. The current document reviews this evidence, presenting a view of some degree of shared ancestry that may have implications for pathophysiology and cell biology. [Copyright &y& Elsevier]

Abstract: Background: Increased circulating endothelial cells (CECs, reflecting endothelial damage) in acute coronary syndromes (ACS) has been reported. However, the inter-relationships of indices of endothelial damage/injury with development of vascular (dys)function, plasma levels of tissue factor (TF, an index of coagulation) and interleukin-6 (IL-6, a pro-inflammatory cytokine) have not been investigated in ACS. We hypothesized that increased CECs can be related to impaired flow-mediated vasodilatation (FMD, an index of endothelial dysfunction) and elevated plasma von Willebrand factor (vWf, also marking endothelial damage/dysfunction), TF and IL-6 in patients with ACS. Methods: We studied 120 patients with ACS (80 acute myocardial infarction and 40 unstable angina; 86 male, age 65±12 years) and 40 matched patients with stable CAD and 40 healthy controls (HC) in a cross-sectional analysis. Plasma vWf, TF and IL-6 levels were measured by ELISA. CECs were quantified using epifluorescence microscope after immunomagnetic separation with CD146. Brachial artery FMD was assessed in a subset of 39 ACS patients. Results: ACS patients had significantly higher CECs, vWf, TF and IL-6 levels, but lower FMD, when compared to stable CAD and HC (all p <0.001) and all were inter-correlated significantly. In ACS, CECs was strongly correlated with FMD (r =−0.64, p <0.001) and TF (r =0.7, p <0.001). In stable CAD, significant correlations were again found between many indices, but on multivariate analysis, IL-6 and vWf were both independently related to FMD. Conclusions: Increased CECs in ACS patients are closely associated with endothelial damage/dysfunction (vWf and FMD), coagulation (TF) and inflammation (IL-6). These inter-relationships support the concept of a central role of endothelial damage/injury in the activation of vascular and coagulation abnormalities in ACS. [Copyright &y& Elsevier]

Abstract: Hyperglycemia has been linked to increased oxidative stress, a resultant endothelial cell dysfunction, and, ultimately, apoptosis. Heme oxygenases (HO-1/HO-2) and the products of their activity, biliverdin/bilirubin and carbon monoxide (CO), play a physiological role in the vascular system. The effects of heme-mediated HO-1 induction, CO, and biliverdin on urinary 8-epi-isoprostane PGF2α and endothelial cell sloughing were examined in an animal model of streptozotocin (STZ)-induced diabetes. Hyperglycemia itself did not affect HO-1 and HO-2 protein levels, but caused a net decrease in HO activity. Weekly heme administration induced HO-1 protein, as demonstrated by immunohistochemistry and Western blot analyses. Administration of biliverdin or the CO donor, CORM-3, decreased urinary 8-epi-isoprostane PGF2α, P < 0.5 compared to diabetes. Hyperglycemia increased endothelial cell sloughing; 8.2 ± 0.8 cells/ml blood in control rats vs. 48 ± 4.8 cells/ml blood in diabetic rats (P < 0.05). Heme administration significantly increased endothelial cell sloughing in diabetic rats (98 ± 8.1 cells/ml blood, P < 0.0007) whereas biliverdin modestly decreased endothelial cell sloughing (26 ± 3.5 cells/ml blood, P < 0.003). Administration of CORM-3 to diabetic rats resulted in a significant decrease in endothelial cell sloughing to 21.3 ± 2.3 (P < 0.001). Administration of SnMP to CORM-3 diabetic rats only partially reversed the protective effects of CORM-3 on endothelial cell sloughing from 21.3 ± 2.3 to 29 ± 2.1 cells/ml, thus confirming a direct protective of CO, in addition to the ability of CORM-3 to induce HO-1 protein. These results demonstrate that exogenously administered CO or bilirubin can prevent endothelial cell sloughing in diabetic rats, likely via a decrease in oxidative stress, and thus represents a novel approach to prophylactic vascular protection in diabetes. [Copyright &y& Elsevier]

This paper was aimed to investigate (1) the early marker of endothelial injury in type 2 diabetes, (2) the intrarenal hemodynamics and renal function, and (3) the therapeutic strategy aiming to restore renal function. Fifty patients (35 normoalbuminuric and 15 albuminuric type 2 diabetes) were examined. Blood was collected for determination of circulating vascular endothelial cells (CEC) and the serum was prepared for determination of transforming growth factor beta (TGFβ), ratio of CEC/TGFβ, and soluble vascular cell adhesion molecule. Intrarenal hemodynamics and renal function were also assessed. The results showed that increased number of circulating EC, elevated TGFβ and depleted ratio of CEC/TGFβ were significantly observed. Intrarenal hemodynamic study revealed a hemodynamic maladjustment characterized by preferential constriction of the efferent arteriole, intraglomerular hypertension and reduction in peritubular capillary flow. It was concluded that early marker of endothelial injury is reflected by increasing number of CEC. Such markers correlate with the glomerular endothelial dysfunction associated with hemodynamic maladjustment. Early detection of endothelial injury and appropriate correction of hemodynamic maladjustment by multidrug vasodilators can effectively restore renal function in type 2 diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: We compared profiles of the numbers of circulating endothelial cells (CEC) and the apoptosis-inducing capacity of serum samples on human endothelial cells (hEC) in on-pump and off-pump coronary artery bypass grafting (CABG) patients. Methods: Blood samples from 30 patients undergoing CABG (randomly assigned to two groups: 15 patients off-pump and 15 on-pump (cardiopulmonary bypass, CPB)) were collected after induction of anesthesia (preoperatively), at weaning from CPB/end of bypass grafting (0h), and 1, 6, 12, 24, and 48h afterwards. CEC were isolated with immunomagnetic anti-CD146-coated Dynabeads, and counted in a Nageotte chamber. The apoptosis-inducing activity of serum samples on hEC was examined by a tissue culture assay system. Apoptotic and normal cells were identified using phase contrast/fluorescence microscopy after DNA dye staining. Results: CEC numbers and proportions of apoptotic hEC were significantly elevated during and after surgery in both groups (p <0.01). Compared with the on-pump group, CEC and proportions of apoptotic hEC were significantly lower (p =0.04 and p =0.03, respectively) in patients having CABG performed off-pump. Starting at comparable baseline levels, the mean CEC-number was highest at 6h postoperatively with 81.9ml−1 (range, 44–141) for on-pump patients and 63.3ml−1 (range, 48–105) for off-pump patients. hEC apoptosis peaked also at T4: 16.5±2.8% versus 11.3±2.2%. In both groups, CEC numbers and proportions of endothelial apoptosis were still elevated at 48h after surgery. Conclusion: The number of circulating endothelial cells and apoptotic endothelial cell death are markers of endothelial activation and damage during CABG. This study provides evidence that CABG with the use of CPB in comparison to OPCAB surgery is associated with a significantly more pronounced endothelial response in the immediate postoperative period. [Copyright &y& Elsevier]

Abstract: Background: Circulating endothelial cells (CECs) in the peripheral blood, probably representing the most direct evidence of endothelial cell damage, are increased in myocardial infarction, unstable angina and critical limb ischaemia. As chronic heart failure is also associated with endothelial abnormalities, we hypothesised that CECs are raised in acute heart failure and that they would correlate with plasma indices of endothelial perturbation, that is, von Willebrand factor (vWf) and soluble E-selectin. Methods: We studied 30 patients with acute heart failure (venesected within 24 h of emergency hospital admission), 30 patients with chronic stable heart failure (venesected as out-patients, all patients in sinus rhythm with ejection fraction ≤40%) and 20 healthy controls. CECs were quantified using epifluorescence microscopy after CD146-immunomagnetic separation and phenotyped by streptavidin/biotin immunocytochemistry. Citrated plasma was analysed for soluble E-selectin and vWf by ELISA. Results: Levels of CECs, vWf and soluble E-selectin were significantly higher (all p <0.01) in patients with heart failure compared to controls, with no significant differences between acute and chronic heart failure. CECs correlated with plasma vWf (p <0.0001) and soluble E-selectin (p =0.022) but not ejection fraction or NYHA class. In multiple regression analysis, heart failure was the only independent predictor of raised CECs (p <0.0001). Immunoperoxidase-defined surface expression of CD34, CD45 and CD36 by CECs was <2%, 0% and 8%, respectively. Conclusion: CECs, a possibly heterologous population, may be used as a novel measure of endothelial damage in acute heart failure and may have implications for the thrombotic risk associated with acute and chronic heart failure and prognosis in this condition. [Copyright &y& Elsevier]

Circulating endothelial cells (CECs) as well as bone-marrow-derived endothelial precursor cells (EPC) play an important role in neovascularisation and tumour growth. To study the impact of neoadjuvant chemotherapy on the amounts of CEC and their precursor cells, mature CEC and their progenitors were quantified by flow cytometry in peripheral blood of breast cancer patients during anthracycline and/or taxane based neoadjuvant chemotherapy and subsequent surgery in comparison to age-matched healthy controls. Cell numbers were tested for correlation with serum levels of angiopoietin-2, erythropoietin, endostatin, endoglin, VEGF and sVCAM-1 as well as clinical and pathological features of breast cancer disease. Circulating endothelial cells were significantly elevated in breast cancer patients and decreased during chemotherapy, whereas EPC (CD34+/VEGFR-2+) as well as their progenitor cell population CD133+/CD34+ and the population of CD34+ stem cells increased. Concomitantly with the increase of progenitor cells an increase of VEGF, erythropoietin and angiopoietin-2 was observed. These data suggest that chemotherapy can only reduce the amounts of mature CEC, probably reflecting detached cells from tumour vessels, whereas the EPC and their progenitors are mobilised by chemotherapy. Since this mobilisation of EPC may contribute to tumour neovascularisation an early antiangiogenic therapy in combination with chemotherapy could be beneficial for the success of cancer therapy.British Journal of Cancer (2006) 94, 524–531. doi:10.1038/sj.bjc.6602952 www.bjcancer.com Published online 31 January 2006 [ABSTRACT FROM AUTHOR]

Cancer, a proliferative disease hallmarked by abnormal cell growth and spread, is largely dependent on tumor neoangiogenesis, with evidence of vascular endothelial dysfunction. Novel ways to assess vascular function in cancer include measuring levels of circulating endothelial cells (CEC). Rare in healthy individuals, increased CEC in peripheral blood reflects significant vascular damage and dysfunction. They have been documented in many human diseases, including different types of cancers. An additional circulating cell population are endothelial progenitor cells (EPC), which have the ability to form endothelial colonies in vitro and may contribute toward vasculogenesis. At present, there is great interest in evaluating the role of EPC as novel markers for tumor angiogenesis and drug therapy monitoring. Recently, exocytic procoagulant endothelial microparticles (EMP) have also been identified. CEC, EPC, and EMP research works may have important clinical implications but are often impeded by methodological issues and a lack of consensus on phenotypic identification of these cells and particles. This review aims to collate existing literature and provide an overview on the current position of CEC, EPC, and EMP in cell biology terms and to identify their significance to clinical medicine, with particular emphasis on relationship with cancer. [ABSTRACT FROM AUTHOR]

Objectives: The circulating endothelial cells (CEC) are proposed to be a non-invasive marker of angiogenesis. The level of CEC in peripheral blood (PB) of acute myeloid leukemia (AML) patients has not been investigated prior to this study. We evaluated the count of resting (rCEC), activated (aCEC) and endothelial progenitor cells (CEPC) in the PB of AML and healthy subjects. In addition we correlated the levels of CEC with disease status, known prognostic factors and response to treatment. Methods: CEC were quantified by utilizing four-color flow cytometry procedures in 48 AML patients at the time of diagnosis and 29 healthy controls. Additionally, measurements were again taken after the first course of induction treatment in 12 of the patients. Results: The numbers of aCEC, rCEC and CEPC were significantly higher in the AML patients than in the controls ( P < 0.0001, P < 0.0001 and P < 0.001, respectively). The CEC count was significantly higher in the AML patients with white blood cell count (WBC) >15 G/L, elevated lactic dehydrogenase (LDH) levels and a higher (over median) absolute blasts count (ABC) in PB than in the group with WBC <15 G/L ( P < 0.03), a normal LDH level ( P < 0.03) and a lower (

Summary: We have been studying the molecular and cellular basis of chronic low-dose, frequently administered, metronomic chemotherapy regimens for the treatment of cancer in a variety of preclinical models, including human breast cancer xenografts. The advantages of metronomic–maintenance-type chemotherapy regimens include significantly reduced host toxicity, potentially reduced costs, increased convenience for patients when oral chemotherapy drugs are used, and the possibility of adopting chronic combination therapies involving conventional chemotherapy drugs and cytostatic molecularly targeted therapies. However, a disadvantage is the empiricism associated with determining the optimal biologic dose (OBD). Recently, we have developed a surrogate biomarker approach involving measurement of circulating endothelial progenitor cells (CEPs) in peripheral blood to help determine the OBD of anti-angiogenic drugs or treatments, including metronomic chemotherapy. Using this approach we determined the OBD for different metronomic chemotherapy regimens and then tested the effect of such drugs for the treatment of established, advanced (high volume) and widespread human breast cancer metastases in immunodeficient mice. This treatment strategy, which was maintained for over 6 months, with no breaks, resulted in marked prolongation of survival and was devoid of overt toxicity. These results suggest the possibility of using metronomic chemotherapy regimens as an adjuvant therapy for early-stage disease, including breast cancer, as was demonstrated recently using long-term daily low-dose UFT for the treatment of early-stage resected non-small cell lung cancer or UFT in combination for early stage breast cancer combined with tamoxifen. [Copyright &y& Elsevier]

Increased angiogenesis has been demonstrated to be a significant prognostic factor in many solid tumors. In the oncohematological setting, it has been associated with myelodysplastic syndromes (MDS), chronic myeloid leukemia, acute lymphoid, and myeloid leukemias. Recently, increased circulating endothelial cells (CECs) have been associated with breast cancer and non-Hodgkin lymphoma (NHL). Based on these premises we analysed total and activated CECs, and endothelial precursors (CEPs) in 50 MDS patients and 20 healthy donors. CECs and CEPs were quantified by flow cytometry. CEC levels were compared with bone marrow (BM) microvessel density (MVD). In addition, some angiogenic factors, namely vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and soluble VEGF-Receptor2 (VEGFR2), were tested in the sera from 25 MDS patients. Total, activated CECs and CEPs were significantly increased in MDS when compared to control group (p < 0.0001); whereas in the MDS cases no association was found with French – American – British (FAB), International Prognostic Scoring System (IPSS) subtypes or survival. Patients with higher CECs also showed higher MVD. Among the cytokines analysed, sVEGFR2 was significantly higher in the lower IPSS risk classes, while the levels of bFGF directly correlated with total and activated CECs. Taken together these data strengthen the hypothesis of a possible role of angiogenesis in MDS pathogenesis. [ABSTRACT FROM AUTHOR]

Shedding of endothelial protein C receptor contributes to vasculopathy and renal injury in lupus: In vivo and in vitro evidence. Background. Candidate biomarkers for vasculopathy in systemic lupus erythematosus (SLE) include circulating endothelial cells and the recently identified endothelial protein C receptor (EPCR) which, when shed, promotes a thrombotic diathesis. This study sought correlation between plasma levels of soluble EPCR and disease manifestation/severity, with a focus on lupus nephritis. Methods. In 81 SLE patients (evaluated cross-sectionally and longitudinally) and 59 healthy controls, levels of soluble EPCR and soluble E-selectin were assessed by sandwich enzyme-linked immunosorbent assay (ELISA), circulating endothelial cells isolated by immunomagnetic separation, and EPCR gene polymorphisms determined. Mechanisms of vascular injury were addressed in vitro in human aortic endothelial cells (HAEC) cultured in the presence and absence of interferon-γ (IFN-γ). Results. The mean level of soluble EPCR was significantly higher in SLE patients (263± 13 ng/mL) than controls (174± 11 ng/mL) ( P< 0.0001). Patients with active or past renal involvement had significantly higher mean soluble EPCR levels (306± 21 ng/mL) ( N= 40) than patients without nephritis (228± 14 ng/mL) ( N= 41) ( P= 0.0033). Mean soluble EPCR correlated positively with serum creatinine ( R= 0.3429, P< 0.0001). The prevalence of the enhanced-shedding EPCR polymorphism A6936G was higher in SLE (41%) ( N= 27) than controls (7%) ( N= 29) ( P= 0.0039). Patient and control plasma were also interrogated for soluble E-selectin, a comparator plasma marker. The results suggest that soluble E-selectin and soluble EPCR are not equivalent end points of vasculopathy and endothelial perturbation in SLE. Although in SLE patients the absence or diminished expression of membrane EPCR on circulating endothelial cells varied, the rare circulating endothelial cells detected in controls invariably expressed membrane-bound EPCR. IFN-γ–treated HAEC expressed less membrane-bound EPCR[133 relative fluorescence units (rfu)] than untreated HAEC (275 rfu); more soluble EPCR was detected in IFN-γ–treated (1.1 ng/106 cells) than untreated HAEC (0.65 ng/106 cells) ( P= 0.027). Conclusion. The results obtained from this cross-sectional/longitudinal study support the hypothesis that the vascular dysfunction characteristic of SLE may be related to a dramatically altered distribution of EPCR, both soluble and membrane-bound forms. [ABSTRACT FROM AUTHOR]

Circulating endothelial cells are associated with future vascular events in hemodialysis patients.Background.Endothelial dysfunction and injury are thought to have a key role in the pathogenesis of cardiovascular disease. We hypothesized that the presence of circulating endothelial cells, as a reflection of ongoing endothelial injury, might provide a novel means for predicting cardiovascular events in hemodialysis subjects who are known to be at marked increased risk for cardiovascular disease.Methods.Circulating endothelial cell number was determined in 29 hemodialysis patients who were then followed for vascular events for 470± 172 days. In a second cohort of 44 hemodialysis patients, circulating endothelial cell number was correlated with markers of inflammation, namely high sensitivity C-reactive protein (hs-CRP), interleukin (IL)-6, IL-10, and monocyte chemoattractant protein-1 (MCP-1), and endothelial dysfunction, soluble vascular cellular adhesion molecule-1 (VCAM-1).Results.Seven of the 19 subjects with elevated circulating endothelial cells (defined as>19 cells per mL) had cardiovascular (N= 5) or vascular (N= 5) events during follow-up, whereas no events occurred in subjects with a low number of circulating endothelial cells (≤19 CECs per mL) (P= 0.04 by Fisher Exact Test). In the second cohort, the number of circulating endothelial cells was independent of all markers of inflammation and endothelial dysfunction.Conclusion.In this hemodialysis population, an increase in circulating endothelial cells was found to predict the development of cardiovascular and vascular events, and to be independent of other known markers of inflammation or endothelial dysfunction. These studies suggest that circulating endothelial cells may be a novel way to assess endothelial health and cardiovascular risk. Further studies to investigate the utility of circulating endothelial cells in predicting cardiovascular risk are needed. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Patients with diabetes mellitus are well known to be at high risk for vascular disease. Circulating endothelial cells (CECs) have been reported to be an ex vivo indicator of vascular injury. We investigated the presence of CECs in the peripheral blood of 25 patients with diabetes mellitus and in nine nondiabetic control donors. Methods: Endothelial cells were isolated from peripheral blood with anti-CD-146-coated immunomagnetic Dynabeads, and were stained with acridine orange dye and counted by fluorescence microscopy. The cells were also stained for von Willebrand factor and Ulex europaeus lectin 1. Results: Patients with diabetes mellitus had an elevated number of CECs (mean 69±30 cells/mI, range 35-126) compared with healthy controls (mean 10±5 cells/ml, range 3-18) (p<0.001). The increase in CECs did not correlate with the levels of HbA1c. Circulating endothelial cell numbers were elevated regardless of glucose levels, suggesting that, even with control of glucose levels, there is increased endothelial cell sloughing. Conclusions: Our study suggests that the higher number of CECs in patients with type 2 diabetes may reflect ongoing vascular injury that is not directly dependent on glucose control. [ABSTRACT FROM AUTHOR]

Heme oxygenase (HO)-1 represents a key defense mechanism against oxidative injury. Hyperglycemia produces oxidative stress and various perturbations of cell physiology. The effect of streptozotocin (STZ)-induced diabetes on aortic HO activity, heme content, the number of circulating endothelial cells, and urinary 8-epi-isoprostane PGF2α (8-Epi) levels in control rats and rats overexpressing or underexpressing HO-1 was measured. HO activity was decreased in hyperglycemia rats. Hyperglycemia increased urinary 8-Epi, and this increase was augmented in rats underexpressing HO-1 and diminished in rats overexpressing HO-1. The number of detached endothelial cells and O2- formation increased in diabetic rats and in hyperglycemia animals underexpressing HO-1 and decreased in diabetic animals overexpressing HO-1 compared with controls. These data demonstrate that HO-1 gene transfer in hyperglycemia rats brings about a reduction in O2 production and a decrease in endothelial cell sloughing. Upregulation of HO-1 decreases oxidant production and endothelial cell damage and shedding and may attenuate vascular complications in diabetes. [ABSTRACT FROM AUTHOR]

Numbers of circulating endothelial cells (CECs) are increased in cancer patients with progressive disease. Also, cancer patients have an increased risk for thrombotic events, being negatively associated with prognosis. Tissue factor (TF), the physiological initiator of coagulation, is present on the surface of many extravascular cells. In 34 samples from cancer patients and in seven from volunteers, CECs were quantified (with endothelium-specific anti-CD146 beads), and TF-activity assessed with a chromogenic assay. All samples displayed very limited TF-activity (patients: 1.6±3.1μU; volunteers: 0.94±1.7μU FXa/100 CECs, P=0.30 by Mann–Whitney test). After in vitro TNFα-stimulation, CECs from both cancer patients and volunteers showed substantially increased TF-activity (endogenous activity: 17.3±6.4μU; after TNFα-stimulation: 73.8±34.3μU FXa; P=0.028, Wilcoxon signed ranks test), reflecting the potential of CECs to generate biologically active TF. As the chromogenic assay determines a mean cellular TF-activity, we also analyzed immunohistochemical TF-antigen expression on CEC subsets. TF-antigen expression was undetectable. CECs isolated from cancer patients do not express TF. CECs can generate functional TF after stimulation and may therefore play a role in (intratumoral) coagulation induction and tumor angiogenesis. [Copyright &y& Elsevier]

Circulating endothelial cells have been established as markers of vascular disease, such as small vessel vasculitis, acute vascular rejection in renal transplant recipients, and cyclosporine-induced endothelial damage. Enumeration of these cells by immunomagnetic isolation and acridine staining remains the gold standard but necessitates considerable experience and expenditure. A simpler test would therefore be of great utility. Hence, our aim was to develop an improved simple assay to enumerate endothelial cells in peripheral blood. We had already used various surface markers to corroborate the endothelial origin of cells. Here, we studied the enumeration of cell numbers with immunomagnetic isolation and a variety of subsequent stains, such as CD31, von Willebrand’s factor (vWF) immunocytochemistry, and Ulex europaeus lectin-1 (UEA-1). Eventually, we devised a simple protocol for enumeration using immunomagnetic isolation and a subsequent UEA-1 lectin stain. We evaluated the use of this protocol in parallel to immunomagnetic isolation and acridine counting alone in 92 renal transplant recipients who underwent renal biopsy. Recovery of various concentrations of human umbilical vein endothelial cells from blood was also studied. Immunomagnetic isolation and subsequent UEA-1 staining permits easier enumeration of circulating endothelial cells in peripheral blood. The assay is simple and easy to use, thus allowing for a more widespread use of circulating endothelial cells as a marker of vascular damage. [ABSTRACT FROM AUTHOR]

Heme oxygenase-1 (HO-1) represents a key defense mechanism against oxidative injury. Hyperglycemia has been linked to increased oxidative stress, leading to endothelial dysfunction, delayed cell replication, and enhanced apoptosis. The effect of streptozotocin (STZ)-induced diabetes on HO activity, HO-1 promoter activity, superoxide anion (O⋅−2, and the number of circulating endothelial cells was measured. The expression of HO-1/HO-2 protein was unchanged, but HO activity was decreased in aortas of diabetic rats compared with control (p<0.05). High glucose decreased HO-1 promoter activity (p<0.05). Hyperglycemia increased O⋅−2 and this increase was augmented with HO-1 inhibition and diminished with HO-1 upregulation (p<0.05). Circulating endothelial cells were significantly higher in diabetic rats and were decreased or increased with administration of the HO-1 inducer (CoPP) or inhibitor (SnMP), respectively (p<0.05). In conclusion, HO-1 upregulation in diabetic rats brings about an increase in serum bilirubin, a reduction in O⋅−2 production, and a decrease in endothelial cell sloughing. [Copyright &y& Elsevier]

Background Increased numbers of CD146-defined circulating endothelial cells (CECs), as are present in the peripheral blood of patients suffering acute coronary syndromes, imply injury to the endothelium. Endothelial damage can also be assessed by the measurement of plasma levels of von Willebrand factor (vWf). Increased levels of procoagulant plasma tissue factor (TF), arising from monocytes/macrophages and endothelial cells, is present in atherosclerosis. We hypothesised increased CECs in patients with ischaemic rest pain (IRP) of the lower limb due to peripheral atherosclerosis and comparable to that seen in patients with acute myocardial infarction (AMI), when compared to patients with intermittent claudication (IC) or healthy controls that would correlate with vWf and TF. [ABSTRACT FROM PUBLISHER]

summary Recent reports have demonstrated that circulating endothelial cells (CECs) are observed in several diseases with vascular injury. Because Kawasaki disease (KD) is one type of systemic vasculitis, we hypothesized that an increased number of CECs may be associated with the appearance of complicated coronary artery lesions (CAL). In the present study we investigated the enumeration and origin of CECs in 20 patients with KD, using an immunohistochemical method with monoclonal antibodies: clone P1H12 against ECs and clone AC133 against endothelial progenitor cells (EPCs), which were derived from the bone marrow. The mean number of CECs increased significantly (P < 0·05) from the acute through the subacute phases of KD compared with both the convalescent phase of KD and healthy children. The mean number of CECs was significantly (P < 0·05) higher in six KD patients with CAL than in 14 KD patients without CAL. The population of EPCs in the total CECs in KD was 4·4 ± 1·2% (range 0–18%). The number of EPCs during the subacute phase was also significantly higher (P < 0·05) in KD patients with CAL than in those without CAL. Our findings indicate that the number of CECs increase in KD vasculitis and suggest that the increased numbers of CECs and EPCs may reflect the EC damage of this disease. [ABSTRACT FROM AUTHOR]

Abstract: The endothelial contribution to vascular disorders has been widely documented in experimental models. However, its implication in human pathology is difficult to investigate, owing to the paucity of non‐invasive methods and of specific endothelial markers. The enumeration of circulating endothelial cells (CEC) released in peripheral blood after vascular injury represents a direct exploration of the endothelium. For this purpose, we have produced a monoclonal antibody (S‐Endo 1), which recognizes CD 146, a molecule expressed on all types of human endothelial cells but absent from haemopoietic cells. Using this antibody, we have designed a specific and sensitive immunocapture test, which allowed us to detect high numbers of CEC in thrombotic, infectious or immunological disorders, while CEC were found to be very rare (<3/ml) in normal subjects. This quantitative approach using CEC might prove useful as a marker of vascular wall injury. Their enumeration is of interest in the clinical follow‐up of vascular disorders, in the evaluation of therapeutic effectiveness or in the direct diagnosis of infectious diseases involving intra‐endothelial microbial agents. Furthermore, an immunological and/or functional study of CEC could allow one to assess their procoagulant and proadhesive properties, as well as their viability, opening new perspectives for CEC investigation in vascular pathology. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the correlation between CEC count and endothelial function, disease activity, and organ involvement in patients with SLE. Background: Premature atherosclerosis in patients with systemic lupus erythematosus (SLE) is not explained by traditional risk factors. Circulating endothelial cells (CEC) have been identified as a surrogate marker of endothelial dysfunction Methods: The present study included 30 premenopausal women with SLE and 20 age and sex matched healthy controls (HC). Endothelial function was studied by flow mediated vasodilation (FMD%) in the brachial artery. Serum levels of VCAM-1, ICAM-1 were measured. Results: FMD% was lower in patients with SLE than HC (3.5 ± 0.4 vs 9.7 ± 3.2, p

A role of endothelial cells (ECs) in Primary Myelofibrosis (PMF) was supposed since JAK2 mutation was found in endothelial precursor cells (EPCs) and in ECs captured by laser microdissection. By Cell Search method, the circulating endothelial cells (CECs) from 14 PMF patients and 5 healthy controls have been isolated and compared by NGS with CD34+Hematopoietic stem and progenitors cells (HSPCs) for panel of 54 myeloid-associated mutations. PMF patients had higher levels of CECs. No mutation was found in HSPCs and CECs from controls, while CECs from PMF patients presented several somatic mutations. 72% of evaluable patients shared at least one mutation between HSPCs and CECs. 2 patients shared the JAK2 mutation, together with ABL1, IDH1, TET2 and ASXL1, KMT2A, respectively. 6 out of 8 shared only NON MPN-driver mutations: TET2 and NOTCH1 in one case; individual paired mutations in TP53, KIT, SRSF2, NOTCH1 and WT1, in the other cases. In conclusion, 70% of PMF patients shared at least one mutation between HSPCs and CECs. These latter harbored several myeloid-associated mutations, besides JAK2V617F mutation. Our results support a primary involvement of EC in PMF and provide a new methodological approach for further studies exploring the role of the "neoplastic" vascular niche. [ABSTRACT FROM AUTHOR]

Circulating endothelial cells (CECs) are viable, apoptotic or necrotic cells, identified by CD 146 surface antigen expression, considered a biomarker of thrombotic risk, given their active role in inflammatory, procoagulant and immune processes of the vascular compartment. Growing evidence establishes that CECs are also involved in the pathogenesis of several hematological and solid malignancies. The primary aim of this study was to verify if CEC levels could predict both the course and treatment responses of splanchnic vein thrombosis (SVT), either in patients affected by myeloproliferative neoplasms (MPNs) or liver disease. Thus, a retrospective multicenter study was performed; fifteen patients receiving anticoagulant oral treatment with vitamin k antagonists (VKA) for SVT were evaluated. Nine patients were affected by MPN, and all of them received cytoreduction in addition to anticoagulant therapy; four of these patients had primary myelofibrosis (PMF) and were treated with ruxolitinib (RUX), and one patient with primary myelofibrosis, two patients with essential thrombocythemia (ET), and two patients with polycythemia vera (PV) were treated with hydroxyurea (HU). Six patients affected by liver diseases (three with liver cirrhosis and three with hepatocellular carcinoma) were included as the control group. CECs were assayed by flow cytometry on peripheral blood at specific time points, for up to six months after enrollment. The CEC levels were related to C-reactive protein (CRP) levels, splenic volume reduction, and thrombus recanalization, mainly in MPN patients. In patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC), for which the mechanism of SVT development is quite different, the relationship between CEC and SV reduction was absent. In conclusion, the CEC levels showed a significant correlation with the extent of venous thrombosis and endothelial cell damage in myeloproliferative neoplasm patients with splanchnic vein thrombosis. Although preliminary, these results show how monitoring CEC levels during cytoreductive and anticoagulant treatments may be useful to improve SVT outcome in MPN patients.